Polyketides are widely used in human and veterinary medicine as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents. Understanding how these compounds are biosynthesized is of paramount importance, not only for the practical values of enhancing product yields, discovering new pathways, and engineering the formation of new biosynthetic products, but for the fundamental scientific value of understanding how complex biochemical reactions are programmed in a multistep biosynthetic pathway. Modular polyketide synthases are among the largest and most complex biological catalysts that are known. Using a small suite of biochemical reactions that are repeated in a tightly programmed manner, these synthases carry out the non-templated, assembly line synthesis of some of the most structurally and stereochemically complex natural products. The proposed studies will produce fundamental new biological insights into the central issue of modern molecular biochemistry, the relationship between protein sequence, structure, and function. A combination of chemical, enzymological, protein engineering, and protein structural approaches to elucidate the individual mechanisms and the molecular basis for the stereochemistry and mechanism of ketosynthase-, ketoreductase, and dehydratase-catalyzed reactions that lead to the formation of complex polyketides formed by multimodular polyketide synthases, including both those biochemical assembly lines with integrated acyltransferase domains and discrete, trans-acting AT domains. The core experimental approach exploits the expression and characterization of individual PKS domains and the use of these recombinant proteins, alone or in combination, to establish the intimate biochemical details of substrate recognition, reaction mechanism, and stereospecificity in polyketide biosynthesis. The PKS systems in which these domains are found are responsible for the biosynthesis of a wide range of complex, physiologically active polyketides, including the macrolide antibiotics erythromycin and picromycin, the acyclic antibiotic bacillaene, the polyether antibiotics nanchangmycin and salinomycin, the polyene antifungal agent amphotericin, and the protein phosphatase 2A inhibitor fostriecin. The major objectives of the proposed research will be: 1) Identification of he role of specific ketoreductase domains in the epimerization of methyl groups during the formation of reduced polyketides and determination of the still obscure mechanism of this epimerization using a newly developed equilibrium isotope exchange assay; 2) Elucidation of the recently elucidated role of reductase-inactive ketoreductase domains that control the stereochemistry of unreduced polyketide intermediates; 3) Determination of the mechanism, substrate specificity, and stereochemistry of dehydration reactions catalyzed by dehydratase domains that form cis double bonds.